Molecular characterization of the myxosporean associated with parasitic encephalitis of farmed Atlantic salmon Salmo salar in Ireland

During seasonal epizootics of neurologic disease and mass mortality in the summers of 1992, 1993 and 1994 on a sea-farm in Ireland, Atlantic salmon Sa lmo salar smells suffered from encephalitis associated with infection by a neurotropic parasite. Based on ultrastructural studies, this neurotropic pa rasite was identified as an intercellular presporogonic multicellular devel opmental stage of a histozoic myxosporean, possibly a Myxobolus species. In order to generate sequence data for phylogenetic comparisons to substantia te the present morphological identification of this myxosporean in the abse nce of detectable sporogony, polymerase chain reaction (PCR), Southern blot hybridization, dideoxynucleotide chain-termination DNA sequencing, and in situ hybridization (ISH) were used in concert to characterize segments of t he small subunit ribosomal RNA (SSU rRNA) gene. Oligonucleotide primers wer e created from sequences of the SSU rRNA gene of M. cerebralis and were emp loyed in PCR experiments using DNA extracted from formalin-fixed paraffin-e mbedded tissue sections of brains from Atlantic salmon smelts in which the myxosporean had been detected by light microscopy. Five segments of the SSU rRNA gene of the myxosporean, ranging in length from 187 to 287 base pairs , were amplified, detected by hybridization with sequence-specific probes, and sequenced. Consensus sequences from these segments were aligned to crea te a partial sequence of the SSU rRNA gene of the myxosporean. Assessments of sequence identity were made between this partial sequence and sequences of SSU rRNA genes from 7 myxosporeans, including Ceratomyxa shasta, Hennegu ya doori, M. arcticus, M. cerebralis, M. insidiosus, M. neurobius, and M. s quamalis. The partial SSU rRNA gene sequence from the myxosporean had more sequence identity with SSU rRNA gene sequences from neurotropic and myotrop ic species of Myxobolus than to those from epitheliotropic species of Myxob olus or Henneguya, or the enterotropic species of Ceratomyxa, and was ident ical to regions of the SSU rRNA gene of M. cerebralis. Digoxigenin-labeled oligonucleotide DNA probes complementary to multiple segments of the SSU rR NA gene of M. cerebralis hybridized with DNA of the parasite in histologic sections of brain in ISH experiments, demonstrating definitively that the s egments of genome amplified were from the organisms identified by histology and ultrastructural analysis. Based on sequence data derived entirely from genetic material of extrasporogonic stages, the SSU rDNA sequence identity discovered in this study supports the hypothesis that the myxosporean asso ciated with encephalitis of farmed Atlantic salmon smelts is a neurotropic species of the genus Myxobolus, with sequences identical to those of M. cer ebralis.